The present technical solution provides for a sensitive quantification of active forms of analytes, preferably proteins, as well as for a determination of the ability of tested substances to bind to the active sites of these analytes. Therefore, current approaches to solve both these problems are summarized below.
Today's standard for sensitive and specific determination of proteins (antigens) in biological samples is called “Enzyme-Linked Immuno-Sorbent Assay” (abbr. ELISA), and to a limited extent, also “Western Blot” (abbr. WB). Both methods use the possibilities to prepare (monoclonal or polyclonal) antibody selectively binding a given antigen, and the amount of bound antibody, which is proportional to the amount of antigen in the sample, is converted into a measurable signal. Preparation of such antibodies has become a quite routine and commercially available method in the past two decades. The most versatile and widely used method of in vitro diagnostics of today is the so-called “Sandwich ELISA”, in which the first antibody is immobilized on a solid carrier, the antigen contained in a biological sample is then bound to the antibody, and after washing, the second detection antibody is bound to the antigen (both antibodies must recognize different epitopes on the same antigen). The detection antibody is conjugated with an enzyme and after repeated washing and addition of substrate, e.g. coloured of luminescent product is produced (depending on the choice of enzyme and substrate) whose amount is proportional to the amount of antigen in the sample. There are several ELISA variants, e.g. fluorophore or radionuclide can be conjugated with the detection antibody instead of an enzyme.
For example, prostate specific antigen (abbr. PSA) can be detected at a concentration of 0.008 ng/ml serum using “ultrasensitive” sandwich ELISA (Abbott Diagnostics). Quantification of PSA in the blood serum is now routinely used for the screening of male population for prostate cancer and in particular for monitoring the patient's response to treatment (Catalona et al. 1991, N Engl J Med, p. 1156; Stamey et al. 1987, N Engl J Med, p. 909). The only definitive treatment of prostate cancer is prostate removal; after this procedure PSA disappears from the blood. If surgery fails to remove all tumour tissue, after some time, the concentration of PSA rises again to the detectable limit. After the surgery, over a period of months to years, PSA levels are below the detection limit of today's methods; therefore more sensitive methods could determine the exact prognosis much earlier than existing methods (Lepor et al. 2012, Bju International, p. 1770).
The very sensitive test using ELISA is generally restricted by the presence of so-called interfering heterophilic antibodies in the blood. These may recognize the sandwich antibodies and thereby connect them without the presence of antigen, which leads to false positive results even in such established methods like the quantification of PSA (Henry et al. 2009, Nature Clinical Practice Urology, p. 164; Preissner et al. 2005, Clinical Chemistry, p. 208). Therefore, to reach at least partial removal of such antibodies, it is sometimes necessary to include additional steps in the processing of blood (de Jager et al. 2005, J. Immunol Methods, p. 124), for which a commercial product is used (Scantibodies). It is also appropriate to include controls for measuring the extent of the interference (Bjerner et al. 2005, Clinical Chemistry, p. 9). Commercial kits for the measurement of PSA normally contain blocking agents, which should help to avoid the effects of interfering antibodies; or the two sandwich antibodies do not originate from the same organism, but even so completely reliable results are not secured (Loeb et al. 2009; Preissner et al. 2005, see above).
It is desirable to further increase the sensitivity of today's ELISA methods, in particular for the above mentioned determination of PSA. Due to high expression of PSA in prostate tissue it can be assumed that there will be a number of tumour markers in the blood in substantially lower concentrations than the concentration of PSA, especially in the early stages of the disease. Furthermore, if antibodies as sensitive as antibodies against PSA are not available against a given antigen, the sensitivity of ELISA decreases substantially. Generally, more sensitive detection would be beneficial also for early detection of viral diseases (HIV) or reliable diagnosis of certain bacterial infections (Lyme disease). Increased sensitivity of up to two orders of magnitude while maintaining a simple ELISA format can usually be achieved by conjugating the detection antibody with an oligonucleotide, which is then quantified by the real-time polymerase chain reaction, i.e. quantitative PCR (qPCR for short). Deoxyribonucleic acid (hereinafter DNA) can be conjugated with the antibody by non-covalent interactions of biotin with streptavidin to be used in method called universal immuno-PCR, abbr. iPCR (Ruzicka et al. 1993, Science, p. 698; Zhou et al. 1993, Nucleic Acids Research, p. 6038; EP 2189539) or by covalent bond formed by chemical agents that are commercially available (e.g. Solulink) to be used in method called direct iPCR (Hendrickson et al. 1995, Nucleic Acids Research, p. 522; EP 0544212; EP 0625211). Despite the high sensitivity of iPCR in laboratory conditions, however, a comparable sensitivity cannot be expected when applied in clinical practice, because the iPCR (like sandwich ELISA) is prone to erroneous results caused by the presence of interfering antibodies in the biological matrices, especially in serum and plasma. New ultrasensitive methods applicable without limitation for determination in biological matrices are therefore still needed. Currently used high-throughput screening (HTS) assays for enzyme inhibitors are mostly based on quantification of either substrate/product or displacement of active site probe by the tested substances (Inglese et al. 2007, Nature Chemical Biology, p. 466). Example of the first type of assay would be absorbance measurement of coloured product originating from reaction of malachite green and phosphate, which is liberated by the action of phosphorylases (Gad et al. 2014, Nature, p. 215). The most versatile assays utilize active site probes and detect their displacement from the active site by the tested substances. Typical readouts in these assays are fluorescence or fluorescence polarisation and the measured property differs between bound and unbound state of the probe which makes possible to discriminate between these two states (Inglese et al. 2007, Nature Chemical Biology, p. 466). Despite the high versatility of these assays, they often suffer from low sensitivity of the detection, which requires the use of high probe and enzyme amounts (Alquicer et al. 2012, J Biomol Screen, p. 1030). Consequently, these assays may tend to produce a lot of false negative results, because weaker inhibitors are not able to displace the probe, which is used in a concentration highly above its Kd. For example, if the working probe concentration is 20 times above its Kd and positive result is reported after 50% decline in the fluorescence polarization, only inhibitors with Ki below 50 nmol·l−1 are detected if 1 μmol·l−1 concentration of tested substances is used. Moreover, the signal to background ratio is typically not higher than one order of magnitude and thus only qualitative information about the binding of tested substances is obtained (Inglese et al. 2007, Nature Chemical Biology, p. 466; Gad et al. 2014, Nature, p. 215). Additional issue of these assays is the inability to accurately screen fluorescent or coloured substances since they interfere with assay readout.
Prostate Specific Membrane antigen (PSMA, also known as GCPII) and Carbonic Anhydrase IX (CA-IX) are both enzymes and are known to be markers of certain types of cancer with possible use as diagnostic and prognostic markers which is limited by the lack of accurate and sensitive bioanalytical methods for their quantification (Barve et al. 2014, Journal of Controlled Release, p. 118; Hyrsl et al. 2009, Neoplasma, p. 298). Both proteins are also targets of drug development campaigns. Drugs consisting of toxin conjugated to small molecular inhibitor of both proteins are under evaluation in clinical and preclinical trials with promising results (Haberkorn et al. 2015, Ann Oncol 26, p. ii33; Krall et al. 2014, Angewandte Chemie-International Edition, p. 4231). Additionally, the inhibition of GCPII is beneficial in animal models of several neuropathies (Barinka et al. 2012, Current Medicinal Chemistry, p. 856), whereas the inhibition of CA-IX has suppressive effects on tumor growth in several animal models (Lock et al. 2013, Oncogene, p. 5210). Despite the promising results, better inhibitors for both proteins are still needed as known compounds exhibit several important adverse effects. More specifically, the current GCPII inhibitors are multiply charged and cannot effectively penetrate the blood brain barrier to reach their intended target organ, whereas known CA-IX inhibitors are sulphonamides with unfavorable pharmacological profiles (Supuran 2008, Nature Reviews Drug Discovery, p. 168). The discovery of novel scaffolds inhibiting these enzymes is strongly limited by the absence of accurate screening methods, the only developed assay for GCPII HTS of inhibitors suffers of low sensitivity (Alquicer et al. 2012, J Biomol Screen, p. 1030) and no HTS of inhibitors is available for CA-IX. On the basis of the present invention we were able to develop currently the most sensitive assays for quantification of both enzymes in complex biological matrices as well as first assays for sensitive and accurate screening of inhibitors of both enzymes.